Figure 1.
Blastocyst Cavities Are Partially Derived from Cytoplasmic Vesicles
(A) Time-lapse of a representative embryo expressing a membrane marker undergoing lumen formation (L marks a lumen). t = 0 min when fluid accumulation is first detectable by automatic segmentation. Scale bar, 20 μm.
(B) Time-lapse of the 1st h of fluid accumulation in an embryo expressing a membrane marker (L marks a lumen). t = 0 min when “string of pearls” microluminal structures are observed. Top row is full embryo view (magenta arrowheads highlight cytoplasmic vesicles, scale bar, 20 μm). Bottom row is insets of cell-cell interfaces indicated by magenta boxes in top row highlighting the appearance of “string of pearls”-like microlumina emergence and resolution (scale bar, 10 μm).
(C) Z slices of phalloidin staining showing cortically-localized vesicles in an E3.0 embryo (top, magenta arrowheads highlight individual vesicles, scale bar, 10 μm). Time-lapse of vesicle secretion into intercellular space in a Lifeact-GFP E3.0 embryo (bottom, “C1” marks secreting cell, “C2” marks adjacent cell, scale bar, 2 μm).
(D) Boxplot of volume for lumina in brefeldin A pharmacologically inhibited embryos.
(E) Boxplot of volume for individual vesicles in WT embryos at E3.0 and E3.5, and ATP1 inhibited embryos at E3.0.
(F) Z slices of an E3.0 embryo expressing Lifeact-GFP showing vesicle localization under Atp1 inhibition conditions (top, magenta arrowheads highlight individual vesicles, scale bar, 10 μm). Time-lapse of vesicle secretion into intercellular space under Atp1 inhibition conditions (bottom, “C1” marks secreting cell, “C2” marks adjacent cell, scale bar, 2 μm).
∗∗∗p < 0.001 ∗∗∗∗p < 0.0001. N = number of embryos. n = number of vesicles.
For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3−Q1; upper whisker, Q3 + (1.5 × IQR).